RNAi promotes heterochromatic silencing through replication-coupled release of RNA Pol II

Abstract

Heterochromatin comprises tightly compacted repetitive regions of eukaryotic chromosomes. The inheritance of heterochromatin through mitosis requires RNA interference (RNAi), which guides histone modification during the DNA replication phase of the cell cycle. Here we show that the alternating arrangement of origins of replication and non-coding RNA in pericentromeric heterochromatin results in competition between transcription and replication in Schizosaccharomyces pombe. Co-transcriptional RNAi releases RNA polymerase II (Pol II), allowing completion of DNA replication by the leading strand DNA polymerase, and associated histone modifying enzymes that spread heterochromatin with the replication fork. In the absence of RNAi, stalled forks are repaired by homologous recombination without histone modification.

Figure 1. Transcription and replication of pericentromeric heterochromatin in fission yeast

a. Pericentromeric heterochromatin on Centromere 3. dh (red), dg (green) and imr (magenta) repeats are shown, bordered by tRNA genes (brown). Replication origins (yellow) are found in each repeat. b. Tiling microarrays of K9me2 ChIP (light blue) and clusters of small RNA sequences (dark blue) from wild-type cells. ChIP-seq reads corresponding to poised (S5-P) and elongating (S2-P) RNA polymerase II enriched in dcr1Δ cells relative to WT cells are in black. c. cDNA clones (beige) from dcr1Δ cells. PolyA sites are indicated as vertical lines and correspond to peaks of PolII. Arrows indicate the direction of “Forward” transcription. d. Alignment of probes used in previous studies indicates that regions enriched for PolII (cen-dg) and transcriptional run-on probes (TRO) lie downstream of forward orientation polyA sites

Figure 2. RNA interference and DNA replication restrict RNA polymerase II accumulation and prevent DNA damage

a. Small RNA (blue) and PolII ChIP-seq reads (black) and regions of significant enrichment (blue and red rectangles) from WT and dcr1Δ on the right arm of Centromere 1. b. A replication bubble is shown, initiated at one of the 3 origin homology regions at centromere 1 (yellow boxes). c. Chromatin immunoprecipitation for RNA PolII and Rad22^Rad52 from HU-arrested and released wild-type (dashed lines) and dcr1Δ (solid lines). Cell cycle progression after release from HU block is monitored by septation index, which peaks coincident with S phase. d. Representative parental and non-parental di-type tetrads from crosses between rhp51Δ cells, defective in homologous recombination, and dcr1Δ or ago1Δ.

Figure 3. Replication fork stalling during heterochromatin replication

Replication intermediates in wild-type and mutant cells resolved by 2D gel electrophoresis and probed with the unique DS/E probe from the ura4 transgene within the dg repeat on chromosome 1 (Fig. 2a). (a) A schematic of replication intermediates in 2D gels indicates joint molecules (X), and forks (Y). Junction molecules indicate fork stalling in (b) WT and (c) dcr1Δ mutant cells, and are reduced in (d) mms19Δ, (e) swi6Δ and (f) clr4Δ.

Figure 4. Replication-coupled transcriptional silencing through histone modification and RNAi

a. The Rik1 complex (red octagon) is recruited to heterochromatic replication forks by interactions with methylated histone H3K9me2 and with the leading strand DNA polymerase (Pol ε, green). Swi6 induces origin firing, but collision with RNA polymerase II (orange) stalls replication forks. RNAi releases PolII by processing of pre-siRNA transcripts (red lines) allowing leading strand DNA polymerase to complete DNA replication and the associated Rik1 histone modification complex (red hexagon) to spread histone modification (black circles). b. In the absence of RNAi, origins fire but PolII is not released, stalling replication forks. Stalled PolII signals repair via homologous recombination instead. Recombination could in principle occur with sister chromatids (shown here) or with other copies of the same repeat (not shown). DNA polymerase ε and the associated Rik1 complex are lost along with the replisome, and fail to spread histone modification into neighboring reporter genes.